The process of cryopreservation is well established to store biological material for a wide variety of purposes in different fields of modern biology and biotechnology. These methods follow very similar basic steps:    1. Treatment of the biological material with a solution containing cryoprotective agent(s).    2. The next step comprises freezing of the biological material to subzero temperature.    3. The so prepared biological material is stored—even for very long time periods—at low temperature, for example in liquid nitrogen.    4. Prior to use the biological material is warmed back.    5. The cryoprotective agent(s) is (are) removed from the biological material. In addition, the biological material may require further steps to restore its original viability.
Several approaches has been tried to improve this above-outlined basic protocol, since the process of cryopreservation is harmful to biological material. Approaches to avoid ice formation through the ultra-rapid cooling and warming rates or by gradual depression of the equilibrium freezing point during cooling to −80° C. have not given a proper solution for every field of cryobiology. Attempts were made to improve survival after freezing: at vitrification highly concentrated aqueous solutions of cryoprotective agents supercool to very low temperatures, allowing intracellular vitrification (Rall and Fahy, 1985). Though Fahy et al. (1984) mentioned the possible use of considerably increased hydrostatic pressure as an additional factor that may facilitate vitrification, but also considered that it had few practical consequences in reproductive biology. Other studies report the use of antifreeze proteins (AFPs) which non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling (Baguisi et al., 1987). The toxic effects of the cryoprotectants and the harmful consequences of the osmotic changes are not negligible at any of the described methods.
These procedures, at present, have a varying degree of efficiency for various applications. For example, in case of preserving embryos, the efficiency of cryopreservation ranges from 0 to 80 percent, depending on the species, freezing method, embryonic stage of development (Ishwar, 1996; Van Wagtendonk-De Leeuw, 1995, 1997; Medeiro, 2002; Reubinoff, 2001; Hammitta, 2003; Archer, 2003; Stachecki, 2002, Leibo and Songsasen, 2002). The success rates for the cryopreservation of human ova, being currently a popular issue, are also far from being satisfactory.
Since 1912 it has been known that water undergoes different phases when submitted to hydrostatic pressure at different temperatures (Bridgman, 1911) (FIG. 7). Solutions can be maintained unfrozen even at low subzero temperatures by applying a certain pressure to them (Bridgeman, 1970). High hydrostatic pressure (HHP) was previously used by Nakahashi et al. (2000, 2001) at subzero preservation of rat livers for transplantation in order to reduce cryoinjuries. This approach uses HHP to reduce substantially the freezing point of the culture medium, thus preserving the biological material at subzero temperature without any of the negative effects of cryopreservation. This approach was found unreliable by the present inventors in preserving mouse embryos, as outlined below in examples 2 and 3.
Hydrostatic pressure in the range of 30-50 MPa usually inhibits the growth of various organisms: the initiation of DNA replication is one of the most pressure-sensitive intracellular processes (Abe et al., 1999). The effects vary in severity depending upon the magnitude and duration of compression (Murakami and Zimmerman, 1973). The cell membrane is noted as a primary site of pressure damage (Palou et al., 1997). High hydrostatic pressure treatment can alter the membrane functionality such as active transport or passive permeability and therefore perturb the physico-chemical balance of the cell (Yager and Chang, 1983; Aldridge and Bruner, 1985; Macdonald, 1987; Schuster and Sleytr, 2002). A recent study by Routray et al. (2002) showed that hydrostatic pressure (5 MPa) facilitated the uptake of DMSO in the experiment conducted with eggs and embryos of medaka (Oryzias latipes), though there was a rapid loss in the viability. The physical or biochemical processes at altered pressure conditions are governed by the principle of Le Chatelier: all reactions that are accompanied by a volume decrease speed up considerably (Murakami and Zimmerman, 1973; Welch et al., 1993; Palou et al., 1997). The application of pressure can lead to a population of conformers of proteins, including partially or completely unfolded conformations. Pressure can cause the denaturation of proteins by the combined effects of breakage of intraprotein interactions and release of cavities followed by the binding of water (Schmid at al., 1975; Weber and Drickamer, 1983; Jaenicke, 1991; Gross and Jaenicke, 1994; Silva et al., 2001).
Recent reports state that hydrostatic pressure enhances the production of shock proteins (Welch et al., 1993; Wemekamp-Kamphuis et al., 2002). Studies describe that instabilities caused by sublethal cold shock in the normal protein synthesis in bacteria are overcome by the synthesis of so-called cold-shock proteins (CSPs, HSPs) (Phadtare et al., 1999). CSPs, HSPs are suspected to have many functions such as RNA chaperones (Graumann and Marahiel, 1999) or transcription activators (LaTena et al., 1991); it was assumed that they also play a role in the protection against freezing (Wouters et al., 1999). Further investigations found that the production of CSPs and HSPs are not only induced by cold shock, but by other environmental stresses also. In E. coli, for example, a type of CSP is produced by nutritional stress (Yamanaka et al., 1998). Another trial showed that high hydrostatic pressure treatment provoked the production of certain cold-induced proteins and heat shock proteins (Welch et al., 1993). Other recent reports state that hydrostatic pressure enhances the production of shock proteins (Wemekamp-Kamphuis, et al., 2002). Since cold-shock and high pressure-treatment both increases CSP and HSP levels, trials were conducted about the possibility of cross-protection. Wemekamp-Kamphuis et al. (2002) found that the level of survival after pressurization of cold-shocked Listeria monocytogenes was 100-fold higher than that of the cells growing at 37° C.
While food-microbiologists study the above-mentioned processes in order to kill detrimental microorganisms (Butz and Ludwig, 1986; Wemekamp-Kamphuis et al., 2002; Spilimbergo et al., 2002), the aim of the present invention is to enhance the survival of cryopreserved biological material.
More attention is paid recently to study the role of shock proteins in cryopreservation. Huang et al. (1999) published that a substantial decrease of a shock protein, HSP90, might be associated with a decline in sperm motility during cooling of boar spermatozoa. Wen-Lei et al. (2003) reported that HSP90 in human spermatozoa was decreased substantially after cryopreservation that may result from protein degradation.
As a summary, HSP90, which is induced by high hydrostatic pressure is:                Cytosolic protein        Molecular chaperone, plays an essential role in stress tolerance, protein folding, signal transduction, etc.        Has been shown to possess an inherent ATPase that is essential for the activation of authentic client proteins in vivo (Pearland Prodromou, 2000).        Associated with semen motility:                    Activate nitric oxide synthetase (NOS) (Garcia-Gardena et al., 1998)            Protect cells from reactive oxygen species (ROS) (Fukuda et al., 1996), which increase significantly during the cooling process and impair greatly sperm motility            Involved in ATP metabolism (Prodromou et al., 1997). ATP level is diminished after cold shock, and would not restore later (Watson, 1981)                        HSP 90 decreased substantially together with the decline of sperm motility after cooling boar semen. It was concluded, that HSP 90 might play a crucial role in regulating porcine sperm motility (Huang et al., 1999)        Geldanamycin, a specific HSP 90 inhibitor, significantly reduced the sperm motility of boar semen in a dose-and time dependant manner (Huang et al., 2000).        
HSP90 decreased substantially after cryopreservation in human spermatozoa, together with the sperm motility; the decrease was not due to leaking, but a result of protein degradation (Wen-Lei CAO et al., 2003).
The accumulation of the pressure effects is lethal beyond a certain level: while irreversible changes of some biomolecules take place at higher pressures, at 300 MPa most bacteria and multicellular organisms die. Though tardigrades—in their active state they die between 100 to 200 MPa—can survive up to 600 MPa if they are in a dehydrated ‘tun’ state (Seki and Toyoshima, 1998).
The present inventors surprisingly found that by applying a hydrostatic pressure challenge, and then by following state of the art cryopreservation protocols, the survival of biological material can be improved significantly. In the context of the present invention, the term survival means, inter alia, improved continued in vitro and in vivo development, higher hatching or implantation and birth rates (in case of embryos); higher post thaw motility and/or improved capacity for fertilization (in case of sperm); improved continued in vitro and in vivo development, improved capacity for being fertilized, higher hatching or implantation and birth rates (in case of oocytes). It is appreciated that the term survival may encompass different other functional characteristics depending on the type of other biological material treated.
For this purpose the pressure tolerance of certain types of biological materials was established (see example 1, 5, and 6), followed by the investigation of several state of the art concepts to achieve the aim of improving the survival of pressurized biological material (see examples 2 and 3). Then the present inventors further investigated the effects of pressure treatment on different types of biological material and unexpectedly found the inventive method of pressure challenge to fulfill their objectives.
In this context we must emphasize that the present inventive concept equally applies to many different cryopreservation protocols, and the choice of those is not limited with respect to the invention. The only necessary step to include in the improved protocols is the step of hydrostatic pressure challenge; the parameters of which can be easily optimized by a person skilled in the art when following the teachings of the present description.